( 12 ) United States Patent
`Furfine et al .
`
`US 11,066,458 B2
`( 10 ) Patent No .:
`( 45 ) Date of Patent :
`* Jul . 20 , 2021
`
`US011066458B2
`
`( * ) Notice :
`
`( 60 )
`
`( 54 ) VEGF ANTAGONIST FORMULATIONS
`SUITABLE FOR INTRAVITREAL
`ADMINISTRATION
`( 71 ) Applicant : REGENERON
`PHARMACEUTICALS , INC . ,
`Tarrytown , NY ( US )
`( 72 ) Inventors : Eric Furfine , Concord , MA ( US ) ;
`Daniel Dix , LaGrangeville , NY ( US ) ;
`Kenneth Graham , Pleasant Valley , NY
`( US ) ; Kelly Frye , Mendham , NJ ( US )
`( 73 ) Assignee : REGENERON
`PHARMACEUTICALS , INC . ,
`Tarrytown , NY ( US )
`Subject to any disclaimer , the term of this
`patent is extended or adjusted under 35
`U.S.C. 154 ( b ) by 42 days .
`This patent is subject to a terminal dis
`claimer .
`( 21 ) Appl . No .: 16 / 582,486
`Sep. 25 , 2019
`( 22 ) Filed :
`( 65 )
`Prior Publication Data
`Jan. 16 , 2020
`US 2020/0017572 A1
`Related U.S. Application Data
`Continuation of application No. 16 / 159,269 , filed on
`Oct. 12 , 2018 , now Pat . No. 10,464,992 , which is a
`continuation of application No. 15 / 879,294 , filed on
`Jan. 24 , 2018 , now Pat . No. 10,400,025 , which is a
`continuation of application No. 15 / 095,606 , filed on
`Apr. 11 , 2016 , now Pat . No. 9,914,763 , which is a
`continuation of application No. 14 / 330,096 , filed on
`Jul . 14 , 2014 , now Pat . No. 9,340,594 , which is a
`continuation of application No. 13 / 914,996 , filed on
`Jun . 11 , 2013 , now Pat . No. 8,802,107 , which is a
`continuation of application No. 13 / 329,770 , filed on
`Dec. 19 , 2011 , now Pat . No. 8,481,046 , which is a
`continuation of application No. 12 / 833,417 , filed on
`Jul . 9 , 2010 , now Pat . No. 8,092,803 , which is a
`continuation of application No. 12 / 560,885 , filed on
`Sep. 16 , 2009 , now Pat . No. 7,807,164 , which is a
`division of application No. 11 / 818,463 , filed on Jun .
`14 , 2007 , now Pat . No. 7,608,261 .
`( 60 ) Provisional application No. 60 / 814,484 , filed on Jun .
`16 , 2006 .
`( 51 ) Int . Cl .
`A61K 38/18
`CO7K 14/71
`COZK 19/00
`A61K 9/00
`A61K 9/19
`A61K 38/17
`CO7K 14/47
`A61M 5/178
`A61K 47/26
`
`( 2006.01 )
`( 2017.01 )
`
`A61K 47/02
`A61K 47/10
`( 52 ) U.S. CI .
`CO7K 14/71 ( 2013.01 ) ; A61K 9/0019
`CPC
`( 2013.01 ) ; A61K 9/0048 ( 2013.01 ) ; A61K 9/19
`( 2013.01 ) ; A61K 38/179 ( 2013.01 ) ; A61K
`38/1793 ( 2013.01 ) ; A61K 47/02 ( 2013.01 ) ;
`A61K 47/10 ( 2013.01 ) ; A61K 47/26 ( 2013.01 ) ;
`A61M 5/178 ( 2013.01 ) ; C07K 14/47
`( 2013.01 ) ; C07K 14/4705 ( 2013.01 ) ; CO7K
`2319/00 ( 2013.01 ) ; CO7K 2319/30 ( 2013.01 )
`( 58 ) Field of Classification Search
`None
`See application file for complete search history .
`
`( 56 )
`
`References Cited
`U.S. PATENT DOCUMENTS
`6,100,071 A
`8/2000 Davis - Smyth et al .
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`5/2005 Davis - Smyth et al .
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`8,110,546 B2
`2/2012 Dix et al .
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`5/2016 Furfine
`9,580,489 B2 †
`2/2017 Furfine
`10,464,992 B2
`11/2019 Furfine et al .
`2003/0113316 Al
`6/2003 Kaisheva et al .
`( Continued )
`FOREIGN PATENT DOCUMENTS
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`OTHER PUBLICATIONS
`Fraser , Hamis M. , et al . , “ Single Injections of Vascular Endothelial
`Growth Factor Trap Block Ovulation in the Macaque and Produced
`Prolonged , Dose - Related Suppression of Ovarian Function , ” ( 2004 )
`J. Clin . Endocrin . & Metabol . 90 ( 2 ) : 1114-1122 .
`( Continued )
`
`Primary Examiner Christine J Saoud
`Assistant Examiner
`Jon M Lockard
`( 74 ) Attorney , Agent , or Firm
`Karl Bozicevic ;
`Bozicevic , Field & Francis LLP
`
`-
`
`( 57 )
`ABSTRACT
`Ophthalmic formulations of a vascular endothelial growth
`factor ( VEGF ) -specific fusion protein antagonist are pro
`vided suitable for intravitreal administration to the eye . The
`ophthalmic formulations include a stable liquid formulation
`and a lyophilizable formulation . Preferably , the protein
`antagonist has an amino acid sequence of SEQ ID NO : 4 .
`
`66 Claims , No Drawings
`Specification includes a Sequence Listing .
`
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`( 2006.01 )
`
`Mylan Exhibit 1164
`Mylan v. Regeneron, IPR2021-00881
`Page 1
`
`

`

`US 11,066,458 B2
`Page 2
`
`( 56 )
`
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`2003/0138417 A1
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`7/2003 Kaisheva et al .
`10/2004 Liu et al .
`12/2005 Davis - Smyth et al .
`9/2006 Dix et al .
`4/2008 Wiegand et al .
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`5/2006
`8/2006
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`2002516871
`WO 97/04801
`WO1998045331
`WO 99/62536
`WO2000075319
`WO 2004/091658
`WO 2005/000895
`WO 2005011734
`WO 2005/020972
`WO 2006/047325
`WO 2006/088650
`WO 2006/104852
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`JP
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`WO
`WO
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`OTHER PUBLICATIONS
`Anonymous : “ Lucentis in the treatment of neovascular ( wet ) age
`related macular degeneration ( AMD ) ” , Jan. 1 , 2007 , pp . 1-54 .
`Petition for Inter Partes Review of U.S. Pat . No. 10,464,992 , as
`submitted to the USPTO on Jan. 7 , 2021 , in Inter Partes Review No.
`IPR2021-00402 , 59 pgs .
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`USPTO on Jan. 7 , 2021 , in Inter Partes Review No. IPR2021-00402 ,
`124 pgs .
`Declaration of Dr. Reiner Gentz , as submitted to the USPTO on Jan.
`7 , 2021 , in Inter Partes Review No. IPR2021-00402 , 76 pgs .
`Fraser et al . , " Single Injections of Vascular Endothelial Growth
`Factor Trap Block Ovulation in the Macaque and Produce a Pro
`longed , Dose - Related Suppression of Ovarian Function , " J. Clin .
`Endocrinol . & Metab . , 90 ( 2 ) : 1114-1122 ( 2004 ) .
`Wulff et al . , “ Prevention of Thecal Angiogenesis , Antral Follicular
`Growth , and Ovulation in the Primate by Treatment with Vascular
`Endothelial Growth Factor Trap R1R2 , ” Endocrinology , 143 ( 7 ) : 2797
`2807 ( 2002 ) .
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`22 , 2011 , as submitted to the USPTO dated Jan. 7 , 2021 , in Inter
`Partes Review No. IPR2021-00402 , 4 pgs .
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`Frye , and Susan Kautz in Support of Response to Office Action in
`U.S. Appl . No. 12 / 835,065 , filed Nov. 22 , 2011 , as submitted to the
`USPTO on Jan. 7 , 2021 , in Inter Partes Review No. IPR2021-00402 ,
`11 pgs .
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`Resume of Reiner Gentz , Ph.D. , as submitted to the USPTO on Jan.
`7 , 2021 , in Inter Partes Review No. IPR2021-00402 , 3 pgs .
`Rudge et al . , “ VEGF Trap as a Novel Antiangiogenic Treatment
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`Review No. IPR2021-00402 , 2 pgs .
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`00402 , 2 pgs .
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`† cited by third party
`
`Mylan Exhibit 1164
`Mylan v. Regeneron, IPR2021-00881
`Page 2
`
`

`

`10
`
`15
`
`25
`
`US 11,066,458 B2
`
`5
`
`1
`VEGF ANTAGONIST FORMULATIONS
`SUITABLE FOR INTRAVITREAL
`ADMINISTRATION
`
`2
`mulations are provided that comprise a VEGF “ trap ” antago
`nist with a pharmaceutically acceptable carrier . In specific
`embodiments , liquid and lyophilized formulations are pro
`vided .
`CROSS - REFERENCE TO RELATED
`In a first aspect , a stable liquid ophthalmic formulation of
`APPLICATIONS
`a VEGF - specific fusion protein antagonist is provided , com
`prising a fusion protein that comprises a receptor component
`This application is a continuation application of U.S.
`consisting essentially of an immunoglobulin - like ( Ig )
`patent application Ser . No. 14 / 330,096 , filed Jul . 14 , 2014 ,
`domain 2 of a first VEGF receptor and Ig domain 3 of a
`which is a continuation of U.S. patent application Ser . No.
`second VEGF receptor , and a multimerizing component
`13 / 914,996 , filed Jun . 11 , 2013 , which issued as U.S. Pat .
`( also termed a “ VEGF trap ” ) . In a specific embodiment of
`No. 8,802,107 on Aug. 12 , 2014 , which is a continuation
`application of U.S. patent application Ser . No. 13 / 329,770 ,
`the VEGF - specific fusion protein antagonist , the first VEGF
`filed Dec. 19 , 2011 , which issued as U.S. Pat . No. 8,481,046
`receptor is Flt1 and the second VEGF receptor is Flkl or
`on Jul . 9 , 2013 , which is a continuation application of U.S.
`Flt4 . In a more specific embodiment the fusion protein has
`patent application Ser . No. 12 / 833,417 , filed Jul . 9 , 2010 ,
`the amino acid sequence of SEQ ID NO : 2 or SEQ ID NO : 4 .
`which issued as U.S. Pat . No. 8,092,803 on Jan. 10 , 2012 ,
`Preferably , the VEGF antagonist is a dimer comprising two
`which is a continuation application of U.S. patent applica
`fusion proteins of SEQ ID NO : 4 .
`tion Ser . No. 12 / 560,885 , filed Sep. 16 , 2009 , which issued
`In one aspect , a stable liquid ophthalmic formulation is
`as U.S. Pat . No. 7,807,164 on Oct. 5 , 2010 , which is a
`divisional application of U.S. patent application Ser . No. 20 provided that comprises 1-100 mg / ml VEGF - specific fusion
`11 / 818,463 , filed Jun . 14 , 2007 , which issued as U.S. Pat .
`protein antagonist , 0.01-5 % of one or more organic co
`No. 7,608,261 on Oct. 27 , 2009 , which claims the benefit
`solvent ( s ) , 30-150 mM of one or more tonicity agent ( s ) ,
`under 35 U.S.C. § 119 ( e ) of U.S. Provisional Application
`5-40 mM of a buffering agent , and optionally , 1.0-7.5 % of
`No. 60 / 814,484 , filed 16 Jun . 2006 , which applications are
`a stabilizing agent , pH between about 5.8-7.0 .
`each hereby incorporated by reference .
`In one or more specific embodiments , the organic co
`solvent may be polysorbate , for example , polysorbate 20 or
`BACKGROUND OF INVENTION
`polysorbate 80 , polyethylene glycol ( PEG ) , for example ,
`PEG 3350 , or propylene glycol , or a combination thereof ;
`Field of the Invention
`the tonicity agent may be , for example , sodium chloride or
`The present invention is directed to pharmaceutical for- 30 potassium chloride ; the stabilizing agent may be sucrose ,
`sorbitol , glycerol , trehalose , or mannitol ; and the buffering
`mulations suitable for intravitreal administration comprising
`agent may be , for example , phosphate buffer . In a specific
`agents capable of inhibiting vascular endothelial growth
`embo ment , the phosphate buffer is a sodium phosphate
`factor ( VEGF ) , and to methods for making and using such
`formulations . The invention includes liquid pharmaceutical
`buffer .
`In various embodiments , the organic co - solvent is poly
`formulations having increased stability , as well as formula- 35
`sorbate and / or PEG , the stabilizing agent is sucrose , the
`tions that may be lyophilize and reconstituted for intravitreal
`buffering agent is phosphate buffer , and the tonicity agent is
`administration .
`sodium chloride .
`More specifically , the stable liquid ophthalmic formula
`Statement of Related Art
`40 tion comprises about 40-50 mg / ml of the VEGF antagonist
`( SEQ ID NO : 4 ) , about 10 mM phosphate buffer , 0.01-3 %
`Vascular endothelial growth factor ( VEGF ) expression is
`polysorbate and / or PEG , 40-135 mM sodium chloride , and
`nearly ubiquitous in human cancer , consistent with its role as
`optionally 5.0 % sucrose , pH about 6.2-6.3 .
`a key mediator of tumor neoangiogenesis . Blockade of
`In a specific preferred embodiment , the stable liquid
`VEGF function , by binding to the molecule or its VEGFR - 2
`receptor , inhibits growth of implanted tumor cells in mul- 45 ophthalmic formulation comprises about 50 mg / ml of the
`tiple different xenograft models ( see , for example , Gerber et
`VEGF antagonist ( SEQ ID NO : 4 ) , 10 mM sodium phos
`al . ( 2000 ) Cancer Res . 60 : 6253-6258 ) . A soluble VEGF-
`phate buffer , 50 mM sodium chloride , 0.1 % polysorbate , and
`specific fusion protein antagonist , termed a " VEGF trap " has
`5 % sucrose , pH about 6.2-6.3 .
`been described ( Kim et al . ( 2002 ) Proc . Natl . Acad . Sci .
`In a specific preferred embodiment , the stable liquid
`USA 99 : 11399-404 ; Holash et al . ( 2002 ) Proc . Natl . Acad . 50 ophthalmic formulation comprises about 50 mg / ml of the
`Sci . USA 99 : 11393-8 ) , which applications are specifically
`VEGF antagonist ( SEQ ID NO : 4 ) , 10 mM sodium phos
`phate buffer , 50 mM sodium chloride , 3 % PEG , and 5 %
`incorporated by reference in their entirety .
`Ophthalmic formulations are known , see for example ,
`sucrose , pH about 6.2-6.3 .
`U.S. Pat . Nos . 7,033,604 and 6,777,429 . An ophthalmic
`In a specific preferred embodiment , the stable liquid
`formulation of a VEGF antibody is described in U.S. Pat . 55 ophthalmic formulation comprises about 40 mg / ml of the
`VEGF antagonist ( SEQ ID NO : 4 ) , 10 mM sodium phos
`No. 6,676,941 .
`Lyophilization ( freeze drying under controlled condi-
`phate buffer , 40 mM sodium chloride , 0.03 % polysorbate ,
`tions ) is commonly used for long - term storage of proteins .
`and 5 % sucrose , pH about 6.2-6.3 .
`The lyophilized protein is substantially resistant to degra-
`In a specific preferred embodiment , the stable liquid
`dation , aggregation , oxidation , and other degenerative pro- 60 ophthalmic formulation comprises about 40 mg / ml of the
`cesses while in the freeze - dried state ( see , for example , U.S.
`VEGF antagonist ( SEQ ID NO : 4 ) , 10 mM sodium phos
`phate buffer , 135 mM sodium chloride , and 0.03 % polysor
`Pat . No. 6,436,897 ) .
`bate , pH about 6.2-6.3 .
`In another aspect , a stable liquid ophthalmic formulation
`BRIEF SUMMARY OF THE INVENTION
`65 is provided that comprises 1-100 mg / ml VEGF - specific
`Stable formulations of a VEGF - specific fusion protein
`fusion protein antagonist ; 0.01-5 % of one or more organic
`co - solvent ( s ) ; 5-40 mM of a buffering agent ; and optionally
`antagonist are provided . Pharmaceutically acceptable for-
`
`Mylan Exhibit 1164
`Mylan v. Regeneron, IPR2021-00881
`Page 3
`
`

`

`US 11,066,458 B2
`
`3
`4
`30-150 mM of one or more tonicity agent ( s ) and / or 1.0-7.5 % 0.01 to 0.15 % of one or more of an organic co - solvent , such
`of a stabilizing agent ; having a pH between about 5.8-7.0 .
`as polysorbate , propylene glycol and / or PEG , and optionally
`In various embodiments , the VEGF antagonist ( SEQ ID
`1-10 % of a stabilizing agent such as sucrose , sorbitol ,
`NO : 4 ) is present at a concentration of about 10 to about 80
`trehalose , glycerol , or mannitol , pH about 5.8-7.0 . In various
`mg / ml . In various embodiments , the VEGF antagonist ( SEQ 5 embodiments , the VEGF antagonist ( SEQ ID NO : 4 ) is
`ID NO : 4 ) is present at a concentration of about 10 , about 20 ,
`present at about 5 , about 10 , about 20 , about 30 , or about 40
`about 30 , about 40 , about 50 , about 60 , about 70 , or about
`mg / ml . In a specific embodiment , the lyophilizable ophthal
`80 mg / ml . In a preferred embodiment , the VEGF antagonist
`mic formulation of the invention comprises 20 mg / ml of the
`( SEQ ID NO : 4 ) is present at a concentration of about 40
`VEGF antagonist , 10 mM sodium phosphate buffer , 0.03 %
`mg / ml .
`10 polysorbate , 0.1 % PEG , and 2.5 % sucrose , pH about 6.2
`In another embodiment , the stabilizing agent is selected
`6.3 . In further embodiments , the lyophilizable formulation
`further comprises sodium chloride . In a specific embodi
`from one or more of sucrose , sorbitol , glycerol , trehalose ,
`ment , the sodium chloride is present at a concentration of
`and mannitol .
`In another embodiment , the organic co - solvent is selected
`about 20 mM . In another specific embodiment , the sodium
`from one or more of polysorbate , for example , polysorbate 15 chloride is present at a concentration of about 67.5 mM .
`20 or polysorbate 80 , polyethylene glycol ( PEG ) , for
`In another specific embodiment , the lyophilizable oph
`example , PEG 3350 , and propylene glycol .
`thalmic formulation of the invention comprises 20 mg / ml of
`In another embodiment , the buffer is a phosphate buffer ,
`the VEGF antagonist , 5 mM sodium phosphate buffer ,
`for example , sodium phosphate .
`0.015 % polysorbate , 20 mM sodium chloride , and 2.5 %
`In another embodiment , the tonicity agent is a salt , for 20 sucrose , pH about 6.2-6.3 .
`In another embodiment , the lyophilizable ophthalmic
`example , sodium chloride .
`In one embodiment , the stable liquid ophthalmic formu-
`formulation comprises 5 mg / ml , 10 mg / ml , or 40 mg / ml
`lation comprises 10 mM sodium phosphate buffer , about
`VEGF antagonist , 5 mM sodium phosphate buffer , 0.015 %
`0.03 to about 0.1 % polysorbate and / or about 3 % PEG or
`polysorbate , 20 mM sodium chloride , and 2.5 % sucrose , at
`propylene glycol , about 40 mM sodium chloride , and about 25 pH 6.2-6.3 . In a specific embodiment , the lyophilizable
`5 % sucrose . In a specific embodiment , the stable liquid
`ophthalmic formulation consists essentially of 5 mg / ml , 10
`ophthalmic formulation comprises 10 mM sodium phos-
`mg / ml , or 40 mg / ml VEGF antagonist ( SEQ ID NO : 4 ) , 5
`phate buffer , about 0.03 % polysorbate , about 40 mM sodium mM sodium phosphate buffer , 0.015 % polysorbate , 20 mM
`chloride , and about 5 % sucrose . In another specific embodi-
`sodium chloride , and 2.5 % sucrose , at pH 6.2-6.3 .
`ment , the pH of the formulation is about 6.2 to about 6.3 . In 30
`In another specific embodiment , the lyophilizable oph
`another specific embodiment , the pH is achieved by mixing
`thalmic formulation comprises 20 mg / ml of the VEGF
`mono- and dibasic sodium phosphate to the desired pH
`antagonist , 5 mM sodium phosphate buffer , 0.015 % poly
`SO te , and 67.5 mM sodium
`pride , pH about 6.2-6.3 . In
`without acid / base titration .
`In a specific embodiment , the stable liquid ophthalmic
`a more specific embodiment , the lyophilizable ophthalmic
`formulation consists essentially of a VEGF antagonist ( SEQ 35 formulation consists essentially of 20 mg / ml of the VEGF
`ID NO : 4 ) at 40 mg / ml , 10 mM sodium phosphate buffer ,
`antagonist ( SEQ ID NO : 4 ) , 5 mM sodium phosphate buffer ,
`polysorbate at 0.03 % , sodium chloride at 40 mm , and
`0.015 % polysorbate , and 67.5 mM sodium chloride , pH
`sucrose at 5 % , pH 6.2-6.3 .
`6.2-6.3 .
`In another aspect , a stable liquid ophthalmic formulation
`In another specific embodiment , the lyophilizable oph
`is provided that comprises about 10 to about 80 mg / ml 40 thalmic formulation comprises 5 mg / ml , 10 mg / ml , or 40
`VEGF antagonist , about 10 mM sodium phosphate buffer ,
`mg / ml VEGF antagonist , 5 mM sodium phosphate buffer ,
`about 0.03 % polysorbate , and about 135 mM sodium chlo-
`0.015 % polysorbate , and 67.5 mM sodium chloride , pH
`about 6.2-6.3 . In a more specific embodiment , the lyophiliz
`ride , pH 6.2 to 6.3 .
`In various embodiments , the VEGF antagonist ( SEQ ID
`able ophthalmic formulation consists essentially of 5 mg / ml ,
`NO : 4 ) is present at a concentration of about 10 to about 80 45 10 mg / ml , or 40 mg / ml VEGF antagonist ( SEQ ID NO : 4 ) ,
`mg / ml . In various embodiments , the VEGF antagonist ( SEQ
`5 mM sodium phosphate buffer , 0.015 % polysorbate , and
`ID NO : 4 ) is present at a concentration of about 10 , about 20 ,
`67.5 mM sodium chloride , pH about 6.2-6.3 .
`about 30 , about 40 , about 50 , about 60 , about 70 , or about
`Generally , the reconstituted formulation is about 2 times
`80 mg / ml . In a specific embodiment , the VEGF antagonist
`the concentration of the pre - lyophilized formulation , e.g. , a
`( SEQ ID NO : 4 ) is present at a concentration of about 40 50 20 mg fusion protein / ml pre - lyophilized formulation is
`reconstituted to a final formulation of 40 mg fusion protein /
`mg / ml .
`In one embodiment , the stable liquid ophthalmic formu-
`ml .
`lation comprises 40 mg / ml of VEGF antagonist ( SEQ ID
`Generally , the lyophilized formulation is reconstituted
`NO : 4 ) , 10 mM sodium phosphate buffer , 0.03 % polysorbate ,
`with sterile water suitable for injection . In one embodiment ,
`and 135 mM sodium chloride at pH 6.2-6.3 . In a specific 55 the reconstitution liquid is bacteriostatic water .
`embodiment , the stable liquid ophthalmic formulation con-
`In another aspect , the invention features a method of
`sists essentially of 40 mg / ml of VEGF antagonist ( SEQ ID
`producing a lyophilized formulation of a VEGF - specific
`NO : 4 ) , 10 mM sodium phosphate buffer , 0.03 % polysorbate ,
`fusion protein antagonist , comprising subjecting the lyo
`philizable formulation of the invention to lyophilization to
`and 135 mM sodium chloride at pH 6.2-6.3 .
`In another aspect , a lyophilizable formulation of a VEGF 60 generate a lyophilized formulation . The lyophilized formu
`antagonist is provided , wherein upon lyophilization fol-
`lation may be lyophilized by any method known in the art
`lowed by reconstitution , a stable liquid ophthalmic formu-
`for lyophilizing a liquid .
`In another related aspect , the invention features a method
`lation as described herein is obtained .
`In another aspect , a lyophilizable formulation of a vas-
`of producing a reconstituted lyophilized formulation of a
`cular endothelial growth factor ( VEGF ) -specific fusion pro- 65 VEGF antagonist , comprising reconstituting the lyophilized
`tein antagonist is provided , comprising 5-50 mg / ml of the
`formulation of the invention to a reconstituted formulation .
`VEGF antagonist , 5-25 mM buffer , such as phosphate buffer ,
`In one embodiment , the reconstituted formulation is twice
`
`Mylan Exhibit 1164
`Mylan v. Regeneron, IPR2021-00881
`Page 4
`
`

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`US 11,066,458 B2
`
`6
`5
`stearate , glycerol monostearate , talc , sodium chloride , dried
`the concentration of the pre - lyophilized formulation , e.g. ,
`skim milk , glycerol , propylene , glycol , water , ethanol and
`the method of the invention comprises : ( a ) producing a
`the like .
`pre - lyophilized formulation of a VEGF - specific fusion pro-
`The term “ lyophilized ” or “ freeze - dried ” includes a state
`tein antagonist , ( b ) subjecting the pre - lyophilized formula-
`tion of step ( a ) to lyophilization ; and ( c ) reconstituting the 5 of a substance that has been subjected to a drying procedure
`such as lyophilization , where at least 90 % of moisture has
`lyophilized formulation of step ( b ) .
`been removed .
`The invention further features ophthalmic formulations
`VEGF Antagonists
`provided in a pre - filled syringe or vial , particularly suitable
`A VEGF antagonist is a compound capable of blocking or
`for intravitreal administration .
`Other objects and advantages will become apparent from 10 inhibiting the biological action of vascular endothelial
`growth factor ( VEGF ) , and includes fusion proteins capable
`a review of the ensuing detailed description .
`of trapping VEGF . In a preferred embodiment , the VEGF
`antagonist is the fusion protein of SEQ ID NO : 2 or 4 ; more
`DETAILED DESCRIPTION OF THE
`preferably , SEQ ID NO : 4 . In specific embodiments , the
`INVENTION
`15 VEGF antagonist is expressed in a mammalian cell line such
`as a CHO cell and may be modified post - translationally . In
`The present invention is not limited to particular methods ,
`a specific embodiment , the fusion protein comprises amino
`and experimental conditions described , as such methods and
`acids 27-457 of SEQ ID NO : 4 and is glycosylated at Asn
`conditions may vary . It is also to be understood that the
`residues 62 , 94 , 149 , 222 and 308. Preferably , the VEGF
`terminology used herein is for the purpose of describing 20 antagonist is a dimer composed of two fusion proteins of
`particular embodiments only , and is not intended to be
`SEQ ID NO : 4 .
`limiting unless indicated , since the scope of the present
`The VEGF antagonist of the methods and formulations of
`invention will be limited only by the appended claims .
`the invention can be prepared by any suitable method known
`Unless stated otherwise , all technical and scientific terms
`in the art , or that comes to be known . The VEGF antagonist
`and phrases used herein have the same meaning as com- 25 is preferably substantially free of protein contaminants at the
`monly understood by one of ordinary skill in the art to which
`time it is used to prepare the pharmaceutically acceptable
`the invention belongs . Although any methods and materials
`formulation . By “ substantially free of protein contaminants ”
`similar or equivalent to those described herein can be used
`is meant , preferably , that at least 90 % of the weight of
`in the practice or testing of the present invention , the
`protein of the VEGF - specific fusion protein antagonist
`preferred methods and materials are now described . All 30 preparation used for making a formulation is VEGF fusion
`protein antagonist protein , more preferably at least 95 % ,
`publications mentioned herein are incorporated herein by
`most preferably at least 99 % . The fusion protein is prefer
`reference .
`ably substantially free of aggregates . “ Substantially free of
`General Description
`Safe handling and administration of formulations com aggregates ” means that at least 90 % of the weight of fusion
`prising proteins represent significant challenges to pharma 35 protein is not present in an aggregate at the time the fusion
`protein is used to prepare the pharmaceutically effective
`ceutical formulators . Proteins possess unique chemical and
`formulation . Unless stated otherwise , the phosphates
`physical properties that present stability problems : a variety
`employed are sodium phosphates and a desired buffering pH
`of degradation pathways exist for proteins , implicating both
`is achieved by mixing appropriate amounts of mono- and
`chemical and physical instability . Chemical instability 40 dibasic sodium phosphate .
`includes deamination , aggregation , clipping of the peptide
`Stable Liquid Ophthalmic Formulations
`backbone , and oxidation of methionine residues . Physical
`In one aspect , the invention provides a stable pharmaceu
`instability encompasses many phenomena , including , for
`tically acceptable formulation comprising a VEGF antago
`example , aggregation and / or precipitation .
`nist , wherein the formulation is a liquid formulation suitable
`Chemical and physical stability can be promoted by 45 for ophthalmic use . Preferably , the liquid formulation com
`prises a pharmaceutically effective amount of the VEGF
`removing water from the protein . Lyophilization ( freeze-
`drying under controlled conditions ) is commonly used for
`antagonist . The formulation can also comprise one or more
`long - term storage of proteins . The lyophilized protein is
`pharmaceutically acceptable carriers , buffers , tonicity
`substantially resistant to degradation , aggregation , oxida-
`agents , stabilizers , and / or excipients . An example of a phar
`tion , and other degenerative processes while in the freeze- 50 maceutically acceptable liquid formulation comprises a
`dried state . The lyophilized protein may be reconstituted
`VEGF antagonist in a pharmaceutically effective amount , a
`with water optionally containing a bacteriostatic preserva-
`buffer , an organic co - solvent such as polysorbate , a tonicity
`agent such as NaCl , and optionally , a stabilizer such as
`tive ( e.g. , benzyl alcohol ) prior to administration .
`sucrose or trehalose .
`Stability is determined in a number of ways at specified
`Definitions
`time points , including determination of pH , visual inspec
`tion of color and appearance , determination of total protein
`The term " carrier ” includes a diluent , adjuvant , excipient ,
`content by methods known in the art , e.g. , UV spectroscopy ,
`or vehicle with which a composition is administered . Car-
`riers can include sterile liquids , such as , for example , water
`and purity is determined by , for example , SDS - PAGE ,
`and oils , including oils of petroleum , animal , vegetable or 60 size - exclusion HPLC , bioassay determination of activity ,
`synthetic origin , such as , for example , peanut oil , soybean
`isoelectric focusing , and isoaspartate quantification . In one
`example of a bioassay useful for determining VEGF antago
`oil , mineral oil , sesame oil and the like .
`The term “ excipient " includes a non - therapeutic agent
`nist activity , a BAF / 3 VEGFR1 / EPOR cell line is used to
`added to a pharmaceutical composition to provide a desired
`determine VEGF165 binding by the VEGF antagonist of the
`consistency or stabilizing effect . Suitable pharmaceutical 65 invention .
`excipients include , for example , starch , glucose , lactose ,
`Liquid formulations can be stored in an oxygen - deprived
`sucrose , gelatin , malt , rice , flour , chalk , silica gel , sodium
`environment . Oxygen - deprived environments can be gener
`
`55
`
`Mylan Exhibit 1164
`Mylan v. Regeneron, IPR2021-00881
`Page 5
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`US 11,066,458 B2
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`Months
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`0
`3
`6
`9
`12
`18
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`24
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`Pass
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`8
`invention belongs . Although any methods and materials
`ated by storing the formulations under an inert gas such as ,
`similar or equivalent to those described herein can be used
`for example , nitrogen or argon . Liquid formulations are
`preferably stored at about 5º C.
`in the practice or testing of the present invention , the
`Ophthalmic Lyophilized Formulations
`preferred methods and material